A novel variant fibrinogen, AαE11del, demonstrating the importance of AαE11 residue in thrombin binding.

INTRODUCTION: We identified a novel heterozygous AαE11del variant in a patient with congenital dysfibrinogenemia. This mutation is located in fibrinopeptide A (FpA). We analyzed the effect of AαE11del on the catalyzation of thrombin and batroxobin and simulated the stability of the complex structure between the FpA fragment (AαG6-V20) peptide and thrombin. MATERIALS AND METHODS: We performed fibrin polymerization and examined the kinetics of FpA release catalyzed by thrombin and batroxobin using purified plasma fibrinogen. To clarify the association between the AαE11 residue and thrombin, we calculated binding free energy using molecular dynamics simulation trajectories. RESULTS: Increasing the thrombin concentration improved release of FpA from the patient's fibrinogen to approximately 90%, compared to the previous 50% of that of normal fibrinogen. Fibrin polymerization of variant fibrinogen also improved. In addition, greater impairment of variant FpA release from the patient's fibrinogen was observed with thrombin than with batroxobin. Moreover, the calculated binding free energy showed that the FpA fragment-thrombin complex became unstable due to the missing AαE11 residue. CONCLUSIONS: Our findings indicate that the AαE11 residue is involved in FpA release in thrombin catalyzation more than in batroxobin catalyzation, and that the AαE11 residue stabilizes FpA fragment-thrombin complex formation.

D-Amino Acids in Peptides from Animals, Including Human: Occurrence, Structure, Bioactivity and Pharmacology.

All life forms typically possess homochirality, with rare exceptions. In the case of peptides and proteins, only L-amino acids are known to be encoded by genes. Nevertheless, D-amino acids have been identified in a variety of peptides, synthesized by animal cells. They include neuroexcitatory and neuroprotective peptides, cardioexcitatory peptides, hyperglycemic hormones, opioid peptides, antimicrobial peptides, natriuretic and defensin-like peptides, and fibrinopeptides. This article is a review of their occurrence, structure and bioactivity. It further explores the pharmacology and potential medical applications of some of the peptides.

Interaction Behaviors of Fibrinopeptide-A and Graphene with Different Functional Groups: A Molecular Dynamics Simulation Approach.

Graphene as a 2-dimentional material has been widely used in the field of biomedical applications. In this study, molecular dynamics simulations are carried out on the fibrinopeptide-A and graphene surfaces with N and O modifications. A new set of parameters for the CHARMM force field are developed to describe the behaviors of the surfaces. Our results indicate that the existence of most oxygen and nitrogen groups may enhance the interaction between the surfaces and the peptide, whereas the substitutional nitrogen on the graphene surface does not make a big difference. The improvement of interaction is not only because of the functional group on the surface, but also the defective morphology. The defective morphology also clears away the surface water layer. Our results suggest that the interactions between graphene biomolecules can be affected by functionalizing the surface with different types of functional groups, which is in accordance with the theory of material design.

Batroxobin binds fibrin with higher affinity and promotes clot expansion to a greater extent than thrombin.

Batroxobin is a thrombin-like serine protease from the venom of Bothrops atrox moojeni that clots fibrinogen. In contrast to thrombin, which releases fibrinopeptide A and B from the NH2-terminal domains of the Aα- and Bß-chains of fibrinogen, respectively, batroxobin only releases fibrinopeptide A. Because the mechanism responsible for these differences is unknown, we compared the interactions of batroxobin and thrombin with the predominant γA/γA isoform of fibrin(ogen) and the γA/γ' variant with an extended γ-chain. Thrombin binds to the γ'-chain and forms a higher affinity interaction with γA/γ'-fibrin(ogen) than γA/γA-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (Kd values of about 0.5 µM) even though it does not interact with the γ'-chain. The batroxobin-binding sites on fibrin(ogen) only partially overlap with those of thrombin because thrombin attenuates, but does not abrogate, the interaction of γA/γA-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a Kd value of 2-5 µM, the α(17-51) and Bß(1-42) regions bind thrombin but not batroxobin. Once bound to fibrin, the capacity of batroxobin to promote fibrin accretion is 18-fold greater than that of thrombin, a finding that may explain the microvascular thrombosis that complicates envenomation by B. atrox moojeni. Therefore, batroxobin binds fibrin(ogen) in a manner distinct from thrombin, which may contribute to its higher affinity interaction, selective fibrinopeptide A release, and prothrombotic properties.

Inferring proteolytic processes from mass spectrometry time series data using degradation graphs.

BACKGROUND: Proteases play an essential part in a variety of biological processes. Besides their importance under healthy conditions they are also known to have a crucial role in complex diseases like cancer. In recent years, it has been shown that not only the fragments produced by proteases but also their dynamics, especially ex vivo, can serve as biomarkers. But so far, only a few approaches were taken to explicitly model the dynamics of proteolysis in the context of mass spectrometry. RESULTS: We introduce a new concept to model proteolytic processes, the degradation graph. The degradation graph is an extension of the cleavage graph, a data structure to reconstruct and visualize the proteolytic process. In contrast to previous approaches we extended the model to incorporate endoproteolytic processes and present a method to construct a degradation graph from mass spectrometry time series data. Based on a degradation graph and the intensities extracted from the mass spectra it is possible to estimate reaction rates of the underlying processes. We further suggest a score to rate different degradation graphs in their ability to explain the observed data. This score is used in an iterative heuristic to improve the structure of the initially constructed degradation graph. CONCLUSION: We show that the proposed method is able to recover all degraded and generated peptides, the underlying reactions, and the reaction rates of proteolytic processes based on mass spectrometry time series data. We use simulated and real data to demonstrate that a given process can be reconstructed even in the presence of extensive noise, isobaric signals and false identifications. While the model is currently only validated on peptide data it is also applicable to proteins, as long as the necessary time series data can be produced.

Proton transfer reactions for improved peptide characterisation.

The combination of deprotonation (via ion/molecule and ion/ion reactions) and low-energy collision-induced dissociation (CID) has been explored for the enhanced characterisation of tryptic peptides via access to different precursor charge states. This approach allows instant access to fragmentation properties of singly and doubly protonated precursors (arising from the availability of mobile protons) in a single experiment. Considering both charge states extended our base of structurally informative data (in comparison with considering just a single charge state) due to generation of additional sequence ions and by obtaining supplementary structural information derived from selective cleavages. Roughly 37% of combined data sets (CID spectra of doubly and singly charged precursor) showed a greater database identification confidence than each set alone. Moreover, comparison between a number of sequence ions of the singly charged precursor and the doubly charged precursor provided a mean of distinguishing the two classes of tryptic peptides (arginine or lysine containing).

Discrimination of isobaric leucine and isoleucine residues and analysis of post-translational modifications in peptides by MALDI in-source decay mass spectrometry combined with collisional cooling.

Collisional cooling employed in an orthogonal time-of-flight mass spectrometer (o-TOF MS) stabilizes fragment ions that are generated by matrix-assisted laser desorption ionization in-source decay (MALDI ISD). By variation of the buffer gas pressure, ISD and "post-source decay" (PSD) dissociation channels can be switched on and off to some extent. Under high-pressure conditions, ISD type fragments of post-translationally modified (PTM) peptides are generated that contain even labile bound side groups; the examples of a phosphorylated and an O-glycosylated peptide are shown. At elevated laser fluences, d- and w-type fragment ions of peptides are detected as a result of high-energy side chain cleavage. This allows for differentiation of the isobaric amino acid residues leucine and isoleucine. Reduction of the cooling efficiency by lowering the buffer gas pressure results in the loss of the d-/w-type species, presumably in secondary metastable dissociation processes. This also enhances cleavage of the side groups from the PTM peptides and can be used to corroborate identification of the modification site. Furthermore, these measuring conditions generate small amounts of fragments containing sequence information about the cyclic part of a disulfide peptide by inducing symmetric and asymmetric cleavage of the intramolecular S-S bond.

Different expression of fibrinopeptide A and related fragments in serum of type 1 diabetic patients with nephropathy.

Type 1 diabetes (insulin-dependent diabetes mellitus, IDDM) is an autoimmune disease affecting about 0.12% of the world's population. Diabetic nephropathy (DN) is a major long-term complication of both types of diabetes and retains a high human, social and economic cost. Thus, the identification of markers for the early detection of DN represents a relevant target of diabetic research. The present work is a pilot study focused on proteomic analysis of serum of controls (n=9), IDDM patients (n=10) and DN patients (n=4) by the ClinProt profiling technology based on mass spectrometry. This approach allowed to identify a pattern of peptides able to differentiate the studied populations with sensitivity and specificity close to 100%. Variance of the results allowed to estimate the sample size needed to keep the expected False Discovery Rate low. Moreover, three peptides differentially expressed in the serum of patients as compared to controls were identified by LC-ESI MS/MS as the whole fibrinopeptide A peptide and two of its fragments, respectively. The two fragments were under-expressed in diabetic patients, while Fibrinopeptide A was over-expressed, suggesting that anomalous turnover of Fibrinopeptide A could be involved in the pathogenesis of DN.

Fragmentation of singly protonated peptides via interaction with metastable rare gas atoms.

The effects of metastable energy level and peptide sequence on the fragmentation patterns of singly charged peptide ions dissociated in collisions with metastable rare gas atoms were studied. Fragmentation spectra of singly charged peptide ions were shown to be more structure-informative and very different from those obtained in low-energy collision-induced dissociation. Unusual odd-electron radical a- and x-ions were observed. Several fragment ions corresponding to a side-chain loss were also observed, which allowed differentiation between leucine and isoleucine. The fragmentation mechanism depends on electronic energy transferred during interaction with metastable gas atoms and proceeds either via Penning ionization with formation of radical odd-electron doubly charged molecular cation or via high-energy excitation of internal degrees of freedom of the peptide cation.

Thrombin induces broad spectrum proteolysis in human serum samples.

BACKGROUND: During clotting, a thrombin cleaves fibrinogen releasing fibrinopeptide A (FPA). FPA is easily identified in serum using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Using MALDI-TOF MS, we observed multiple, progressively shorter fragments of serum FPA. Following ambient incubation of serum, variations in the content of FPA fragments occur over time. Denaturation of a thrombin by heating the serum sample appears to minimize this variation. These observations suggest that intrinsic proteolytic and peptidolytic activity is elevated in serum and perhaps originates from the coagulation cascade enzymes themselves, especially a thrombin. METHODS: Extrinsic addition of a thrombin to a subset (3-30 kDa) of plasma proteins was carried out to induce proteolysis and to examine the resultant peptides to reveal a thrombin susceptible parent proteins. One of these identified proteins, hemopexin, was directly digested by a thrombin and the peptides examined to confirm the observations from the initial plasma protein digestion. RESULTS: Extrinsic addition of a thrombin to a subset (3-30 kDa) of plasma proteins results in wide-spread digestion of proteins unrelated to coagulation, revealing a substrate range encompassing more than fibrinogen. Direct digestion of one of these proteins, hemopexin, by a thrombin confirms these observations. CONCLUSIONS: The resulting peptides indicate broad tolerance beyond the consensus R-G cleavage site of fibrinogen; in fact, there appears to be no bias for the amino acid following the R/K residue. These data support our hypothesis that the enzymatic activities inherent to coagulation, or at least to thrombin, contribute to destabilization of the protein and peptide content of serum.

The action of MBL-associated serine protease 1 (MASP1) on factor XIII and fibrinogen.

The complement system is an important recognition and effector mechanism of the innate immune system that upon activation leads to the elimination of foreign bodies. It can be activated through three pathways of which the lectin pathway is one. The lectin pathway relies on the binding of mannan-binding lectin (MBL) or the ficolins and the subsequent activation of the MBL-associated serine proteases (MASPs), namely, MASP1, 2 and 3 which all form complexes with both MBL and the ficolins. Major substrates have only been identified for MASP2 i.e. C4 and C2. For MASP1 only a few protein substrates which are cleaved at a low rate have been identified while none are known for MASP3. Since chromogenic substrate screenings have shown that MASP1 has thrombin-like activity, we wanted to investigate the catalytic potential of MASP1 towards two major proteins involved in the clotting process, fibrinogen and factor XIII, and compare the activity directly with that of thrombin. We found that rMASP1 and thrombin cleave factor XIII A-chain and the fibrinogen beta-chain at identical sites, but differ in cleavage of the fibrinogen alpha-chain. The thrombin turnover rate of factor XIII is approximately 650 times faster than that of rMASP1 at 37 degrees C, pH 7.4. rMASP1 cleavage of fibrinogen leads to the release of the proinflammatory peptide fibrinopeptide B. Thus rMASP1 has similar, but not identical specificity to thrombin and its catalytic activity for factor XIII and fibrinogen cleavage is much lower than that of thrombin. Nevertheless, rMASP1 can drive the formation of cross-linked fibrinogen. Since MASP1 is activated on contact of MBL or the ficolins with microorganisms, fibrinogen and factor XIII may be involved in the elimination of invading pathogens.

Surface-induced dissociation of peptides and protein complexes in a quadrupole/time-of-flight mass spectrometer.

A novel in-line surface-induced dissociation (SID) device was designed and implemented in a commercial QTOF instrument (Waters/Micromass QTOF II). This new setup allows efficient SID for a broad range of molecules. It also allows direct comparison with conventional collision-induced dissociation (CID) on the same instrument, taking advantage of the characteristics of QTOF instrumentation, including extended mass range, improved sensitivity, and better resolution compared with quadrupole analyzers and ion traps. Various peptides and a noncovalent protein complex have been electrosprayed and analyzed with the new SID setup. Here we present SID of leucine enkephalin, fibrinopeptide A, melittin, insulin chain-B, and a noncovalent protein complex from wheat, heat shock protein 16.9. The SID spectra were also compared to CID spectra. With the SID setup installed, ion transmission proved to be efficient. SID fragmentation patterns of peptides are, in general, similar to CID, with differences in the relative intensities of some peaks such as immonium ions, backbone cleavage b- versus y-type ions, and y- versus y-NH3 ions, suggesting enhanced accessibility to high-energy/secondary fragmentation channels with SID. Furthermore, these results demonstrate that the in-line SID setup is a valid substitute for CID, with potential advantages for activation of singly/multiply charged peptides and larger species such as noncovalent protein complexes.

Trapped-ion cell with improved DC potential harmonicity for FT-ICR MS.

The trapped-ion cell is a key component critical for optimal performance in Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS). To extend the performance of FT-ICR MS, we have developed a new cell design that is capable of generating a DC trapping potential which closely approaches that of an ideal Penning trap, i.e., a 3D axial quadrupolar potential distribution. The new cell design was built upon an open cylindrical geometry, supplemented with two pairs of cylindrical compensation segments. Electric potential calculations for trial cell geometries were aimed at minimizing spatial variations of the radial electric field divided by radius. The resulting cell proportions and compensation voltages delivered practically constant effective ion cyclotron frequency that was independent of ion radial and axial positions. Our customized 12 tesla FT-ICR instrument was upgraded with the new cell, and the performance was characterized for a range of ion excitation power and ion populations. Operating the compensated cell at increased postexcitation radii, approximately 0.7 of the cell inner radius, resulted in improved mass measurement accuracy together with increased signal intensity. Under these same operating conditions the noncompensated open cell configuration exhibited peak splitting and reduced signal life time. Mass accuracy tests using 11 calibrants covering a wide m/z range reproducibly produced under 0.05 ppm RMS precision of the internal calibration for reduced ion populations and the optimal excitation radius. Conditions of increased ion population resulted in a twofold improvement in mass accuracy compared with the noncompensated cell, due to the larger achievable excitation radii and correspondingly lower space charge related perturbations of the calibration law.

Simultaneous fragmentation of multiple ions using IMS drift time dependent collision energies.

Ion mobility spectrometry coupled with mass spectrometry (IMS-MS) was utilized to evaluate an ion collision energy ramping technique that simultaneously fragments a variety of species. To evaluate this technique, the fragmentation patterns of a mixture of ions ranging in mass, charge state, and drift time were analyzed to determine their optimal fragmentation conditions. The precursor ions were pulsed into the IMS-MS instrument and separated in the IMS drift cell based on mobility differences. Two differentially pumped short quadrupoles were used to focus the ions exiting the drift cell, and fragmentation was induced by collision induced dissociation (CID) between the conductance limiting orifice behind the second short quadrupole and before the first octopole in the mass spectrometer. To explore the fragmentation spectrum of each precursor ion, the bias voltages for the short quadrupoles and conductance limiting orifices were increased from 0 to 50 V above nonfragmentation voltage settings. An approximately linear correlation was observed between the optimal fragmentation voltage for each ion and its specific drift time, so a linear voltage gradient was employed to supply less collision energy to high mobility ions (e.g., small conformations or higher charge state ions) and more to low mobility ions. Fragmentation efficiencies were found to be similar for different ions when the fragmentation voltage was linearly ramped with drift time, but varied drastically when only a single voltage was used.

Homophenotypic Aalpha R16H fibrinogen (Kingsport): uniquely altered polymerization associated with slower fibrinopeptide A than fibrinopeptide B release.

We detail for the first time the uniquely altered fibrin polymerization of homophenotypic Aalpha R16H dysfibrinogen. By polymerase chain reaction amplification and DNA sequencing, our new proposita's genotype consisted of a G>A transition encoding for Aalpha R16H, and an 11 kb Aalpha gene deletion. High-performance liquid chromatography disclosed fibrinopeptide A release approximately six times slower than its fibrinopeptide B. Turbidimetric analyses revealed unimpaired fibrin repolymerization, and abnormal thrombin-induced polymerization (1-7 mumol/l fibrinogen, > 96% coagulable), consisting of a prolonged lag time, slow rate, and abnormal clot turbidity maxima, all varying with thrombin concentration. For example, at 0.2-3 U/ml, the resulting turbidity maxima ranged from lower to higher than normal control values. By scanning electron microscopy, clots formed by 0.3 and 3 thrombin U/ml displayed mean fibril diameters 42 and 254% of the respective control values (n = 400). Virtually no such differences from control values were demonstrable, however, when clots formed in the presence of high ionic strength (micro = 0.30) or of monoclonal antibeta(15-42)IgG. The latter also prolonged the thrombin clotting time approximately three-fold. Additionally, thrombin-induced clots displayed decreased elastic moduli, with G' values of clots induced by 0.3, 0.7 and 3 thrombin U/ml corresponding to 11, 34, and 45% of control values. The results are consistent with increased des-BB fibrin monomer generation preceding and during polymerization. This limited the inherent gelation delay, decreased the clot stiffness, and enabled a progressively coarser, rather than finer, network induced by increasing thrombin concentrations. We hypothesize that during normal polymerization these constitutive des-BB fibrin monomer properties attenuate their des-AA fibrin counterparts.

B:b interactions are essential for polymerization of variant fibrinogens with impaired holes 'a'.

BACKGROUND: Fibrin polymerization is mediated by interactions between knobs 'A' and 'B' exposed by thrombin cleavage, and holes 'a' and 'b' always present in fibrinogen. The role of A:a interactions is well established, but the roles of knob:hole interactions A:b, B:b or B:a remain ambiguous. OBJECTIVES: To determine whether A:b or B:b interactions have a role in thrombin-catalyzed polymerization, we examined a series of fibrinogen variants with substitutions altering holes 'a': gamma364Ala, gamma364His or gamma364Val. METHODS: We examined thrombin- and reptilase-catalyzed fibrinopeptide release by high-performance liquid chromatography, fibrin clot formation by turbidity, fibrin clot structure by scanning electron microscopy (SEM) and factor (F) XIIIa-catalyzed crosslinking by sodium dodecylsulfate polyacrylamide gel electrophoresis. RESULTS: Thrombin-catalyzed fibrinopeptide A release was normal, but fibrinopeptide B release was delayed for all variants. The variant fibrinogens all showed markedly impaired thrombin-catalyzed polymerization; polymerization of gamma364Val and gamma364His were more delayed than gamma364Ala. There was absolutely no polymerization of any variant with reptilase, which exposed only knobs 'A'. SEM showed that the variant clots formed after 24 h had uniform, ordered fibers that were thicker than normal. Polymerization of the variant fibrinogens was inhibited dose-dependently by the addition of either Gly-Pro-Arg-Pro (GPRP) or Gly-His-Arg-Pro (GHRP), peptides that specifically block holes 'a' and 'b', respectively. FXIIIa-catalyzed crosslinking between gamma-chains was markedly delayed for all the variants. CONCLUSION: These results demonstrate that B:b interactions are critical for polymerization of variant fibrinogens with impaired holes 'a'. Based on these data, we propose a model wherein B:b interactions participate in protofibril formation.

Ion funnel trap interface for orthogonal time-of-flight mass spectrometry.

A combined electrodynamic ion funnel and ion trap coupled to an orthogonal acceleration (oa)-time-of-flight mass spectrometer was developed and characterized. The ion trap was incorporated through the use of added terminal electrodynamic ion funnel electrodes enabling control over the axial dc gradient in the trap section. The ion trap operates efficiently at a pressure of approximately 1 Torr, and measurements indicate a maximum charge capacity of approximately 3 x 10(7) charges. An order of magnitude increase in sensitivity was observed in the analysis of low concentration peptides mixtures with orthogonal acceleration (oa)-time-of-flight mass spectrometry (oa-TOF MS) in the trapping mode as compared to the continuous regime. A signal increase in the trapping mode was accompanied by reduction in the chemical background, due to more efficient desolvation of, for example, solvent related clusters. Controlling the ion trap ejection time was found to result in efficient removal of singly charged species and improving signal-to-noise ratio (S/N) for the multiply charged analytes.

Role of 'B-b' knob-hole interactions in fibrin binding to adsorbed fibrinogen.

BACKGROUND: The formation of a fibrin clot is supported by multiple interactions, including those between polymerization knobs 'A' and 'B' exposed by thrombin cleavage and polymerization holes 'a' and 'b' present in fibrinogen and fibrin. Although structural studies have defined the 'A-a' and 'B-b' interactions in part, it has not been possible to measure the affinities of individual knob-hole interactions in the absence of the other interactions occurring in fibrin. OBJECTIVES: We designed experiments to determine the affinities of knob-hole interactions, either 'A-a' alone or 'A-a' and 'B-b' together. METHODS: We used surface plasmon resonance to measure binding between adsorbed fibrinogen and soluble fibrin fragments containing 'A' knobs, desA-NDSK, or both 'A' and 'B' knobs, desAB-NDSK. RESULTS: The desA- and desAB-NDSK fragments bound to fibrinogen with statistically similar K(d)'s of 5.8 +/- 1.1 microm and 3.7 +/- 0.7 microm (P = 0.14), respectively. This binding was specific, as we saw no significant binding of NDSK, which has no exposed knobs. Moreover, the synthetic 'A' knob peptide GPRP and synthetic 'B' knob peptides GHRP and AHRPY, inhibited the binding of desA- and/or desAB-NDSK. CONCLUSIONS: The peptide inhibition findings show both 'A-a' and 'B-b' interactions participate in desAB-NDSK binding to fibrinogen, indicating 'B-b' interactions can occur simultaneously with 'A-a'. Furthermore, 'A-a' interactions are much stronger than 'B-b' because the affinity of desA-NDSK was not markedly different from desAB-NDSK.

Direct evidence for specific interactions of the fibrinogen alphaC-domains with the central E region and with each other.

The carboxyl-terminal regions of the fibrinogen Aalpha chains (alphaC regions) form compact alphaC-domains tethered to the bulk of the molecule with flexible alphaC-connectors. It was hypothesized that in fibrinogen two alphaC-domains interact intramolecularly with each other and with the central E region preferentially through its N-termini of Bbeta chains and that removal of fibrinopeptides A and B upon fibrin assembly results in dissociation of the alphaC regions and their switch to intermolecular interactions. To test this hypothesis, we studied the interactions of the recombinant alphaC region (Aalpha221-610 fragment) and its subfragments, alphaC-connector (Aalpha221-391) and alphaC-domain (Aalpha392-610), between each other and with the recombinant (Bbeta1-66)2 and (beta15-66)2 fragments and NDSK corresponding to the fibrin(ogen) central E region, using laser tweezers-based force spectroscopy. The alphaC-domain, but not the alphaC-connector, bound to NDSK, which contains fibrinopeptides A and B, and less frequently to desA-NDSK and (Bbeta1-66)2 containing only fibrinopeptides B; it was poorly reactive with desAB-NDSK and (beta15-66)2 both lacking fibrinopeptide B. The interactions of the alphaC-domains with each other and with the alphaC-connector were also observed, although they were weaker and heterogeneous in strength. These results provide the first direct evidence for the interaction between the alphaC-domains and the central E region through fibrinopeptide B, in agreement with the hypothesis given above, and indicate that fibrinopeptide A is also involved. They also confirm the hypothesized homomeric interactions between the alphaC-domains and display their interaction with the alphaC-connectors, which may contribute to covalent cross-linking of alpha polymers in fibrin.